scholarly journals Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides.

1983 ◽  
Vol 97 (4) ◽  
pp. 1243-1248 ◽  
Author(s):  
A M Tartakoff ◽  
P Vassalli
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Lectin Binding ◽  
Electron Microscopic Level ◽  
Asymmetric Distribution ◽  
Secretory Product ◽  
Electron Microscopic ◽  
Oligosaccharide Structure ◽  
Golgi Stack ◽  
Cellular Components

We investigated the subcellular sites of glycoprotein oligosaccharide maturation by using lectin conjugates to stain lightly-fixed, saponin-permeabilized myeloma cells. At the electron microscopic level, concanavalin A-peroxidase stains the cisternal space of the nuclear envelope, the rough endoplasmic reticulum, and cisternae along the proximal face of the Golgi stack. Conversely, wheat germ agglutinin-peroxidase stains cisternae along the distal face of the Golgi stack, associated vesicles, and the cell surface. These observations confirm the existence of two qualitatively distinct Golgi subcompartments, show that the lectin conjugates can be employed as relatively proximal or distal Golgi markers under conditions of excellent ultrastructural preservation, suggest that the asymmetric distribution of qualitatively distinct oligosaccharides is a property of underlying cellular components and not simply of the principal secretory product, and suggest that the oligosaccharide structure recognized by wheat germ agglutinin is attained during transport from the proximal toward the distal face of the Golgi stack.

scholarly journals Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

1977 ◽  
Vol 25 (11) ◽  
pp. 1187-1200 ◽  
Author(s):  
W D Geoghegan ◽  
G A Ackerman
Keyword(s):  
Horseradish Peroxidase ◽  
Concanavalin A ◽  
Colloidal Gold ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Indirect Detection ◽  
Electron Microscopic Level ◽  
Specific Cell ◽  
Major Protein ◽  
Electron Microscopic

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.

scholarly journals FIXATION OF NEURAL TISSUES FOR ELECTRON MICROSCOPY BY PERFUSION WITH SOLUTIONS OF OSMIUM TETROXIDE

1962 ◽  
Vol 12 (2) ◽  
pp. 385-410 ◽  
Author(s):  
Sanford L. Palay ◽  
S. M. McGee-Russell ◽  
Spencer Gordon ◽  
Mary A. Grillo
Keyword(s):  
Electron Microscopy ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Neural Tissues ◽  
Structural Relations ◽  
Cellular Components

This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

Changing surface antigen and carbohydrate patterns during the development of Oesophagostomum dentatum

Parasitology ◽  
1999 ◽  
Vol 119 (5) ◽  
pp. 491-501 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI ◽  
A. DAUGSCHIES
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Soybean Agglutinin ◽  
Peanut Agglutinin ◽  
Oesophagostomum Dentatum ◽  
Periodate Treatment

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin- labelled lectins. Specific binding of antibodies was observed in all parasitic stages – freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode – infective, invasive, histotropic, and luminal – and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite–parasite interactions is discussed.

scholarly journals Exclusion of Golgi Residents from Transport Vesicles Budding from Golgi Cisternae in Intact Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1263-1270 ◽  
Author(s):  
Lelio Orci ◽  
Mylène Amherdt ◽  
Mariella Ravazzola ◽  
Alain Perrelet ◽  
James E. Rothman
Keyword(s):  
Steady State ◽  
Protein Transport ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Central Feature ◽  
Electron Microscopic ◽  
Anterograde Transport ◽  
Intact Cells ◽  
Golgi Stack ◽  
Transport Vesicles

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.

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